Prevalence, risk-factors, and antimicrobial susceptibility profile of methicillin-resistant Staphylococcus aureus (MRSA) obtained from nares of patients and staff of Sokoto state-owned hospitals in Nigeria

Aim: The aim of the study was to determine the prevalence of methicillin-resistant Staphylococcus aureus (MRSA) obtained from the nasal cavity of participants and investigate the antibiotic resistance profiles of the isolates from Sokoto state, Nigeria. Methods: Nasal swabs of both nares were obtained from 378 participants across three study centers within the six-month study period. The Staphylococcus aureus isolates recovered were characterized, and their resistance phenotype determined in conjunction with MRSA prevalence. Results: Phenotypic screening of isolates obtained in this study revealed a total of 131 (17.3%) coagulase-positive Staphylococci out of 756 samples. Of this number, there were 81 (61.8%) S. aureus, 36 (27.5%) Staphylococcus intermedius, 6 (4.5%) Staphylococcus hyicus, and 8 (6.1%) Staphylococcus schleiferi. Conclusion: This study found a prevalence of 61.8% and 46.9% of S. aureus and MRSA among the studied hospitals in Sokoto state, thus demonstrating that the nares of the hospital populace are not free from S. aureus and MRSA colonization.


Introduction
Staphylococcus aureus is a major cause of morbidity and mortality globally, both within the community and in healthcare settings [1]. Its ability to cause disease is enhanced by its virulence factors and resistance to antibiotics used in its treatment, exemplified by the advent of methicillin-resistant Staphylococcus aureus (MRSA). Most MRSA strains harbor mecA gene on a staphylococcal cassette chromosome mec (SCCmec) and codes for a modified penicillin binding protein (PBP2a). This protein has a reduced affinity for all beta-lactam and Betalactam/beta-lactamase inhibitor combination antibiotics [2]. The burden of MRSA is mostly less than 50% in most of the African countries. In Tunisia, MRSA prevalence is from 16% to 41%, South Africa 24% to 36%, Botswana 23%-44%, Egypt 45% and 52%, Libya 31%) and Nigeria 55% (Northern) and 39% (Southern) [3], [4], [5]. This clear variation might be due to different environmental factors, strain diversity and differences in antibiotics usage. In Kenya, there is a noticeable difference in MRSA prevalence reported in clinical isolates; one study [6] reported a prevalence rate of about 3.7%. while another reported 87.2% [7]. A previous study showed that S. aureus clones obtained from nasal specimens of septicemic patients were identical to those from blood isolates (≈82%), thus evincing the relationship between nasal carriage and infection. Healthy carriers may transmit the organism to other members of the community or immunocompromised persons [8]. With this study, we intended to fill in the knowledge gap about resistance trends of MRSA both in healthcare and community settings in Sokoto, North West Nigeria. The results of the present study will facilitate implementation of strategies for the prevention and effective management of methicillin-resistant infections in Sokoto, Nigeria. This study examines the prevalence of methicillin resistant Staphylococcus aureus (MRSA) obtained from the nasal cavity of study participants and explores the antibiotic resistance profiles of isolates from Sokoto state-owned hospitals.

Study area
This work was carried out in Sokoto State, in the extreme north-west of Nigeria, between longtitudes 4°8¹ and 6°54¹ and latitude 12°N and 13°58¹N. The state includes twenty-three (23) Local Government areas and shares borders with Niger Republic to the north and northeast, Kebbi state, to the west and south-east, and Zam-fara state, to the east. The State covers an area of 32,000 km 2 [9]. The warmest months are February to April, when daytime temperatures can exceed 45°C. The State has a population of 3,696,999 [10]. There are two major ethnic groups, Hausa and Fulani. The majority of the populace are agriculturists, although trades such as blacksmithing, dyeing and leather tanning are also practiced [11].

Ethical consideration
Ethical clearance was obtained from the ethics review boards of Sokoto State Ministry of Health and three hospitals spread across three local government areas in the state (Maryam Abacha Women's and Children's Hospital, Specialist Hospital and Orthopedic Hospital Wamakko) after approval of the research protocol (Approval num-ber=SMH/1580/V. IV).

Informed consent
The participants in the study gave informed consent following detailed (oral) explanation of the study and assurance of anonymity.

Sample size determination
The sample size was determined using the formula specified by Thrusfield et al. [12] by taking a previous prevalence of S. aureus, 40.6% [13], found in a study carried out to establish the prevalence and nasal carriage of MRSA among adult patients in Cameroon. Accordingly, the calculated value for sample size equaled 232 samples.

Exclusion criteria
We excluded participants with nasal anatomical deformities or bleeding nostrils from the study.

Specimen collection
Three health-care centers from three of the largest local government areas (LGA) in Sokoto state, specifically Sokoto South (Specialist Hospital Sokoto), Sokoto North (Maryam Abacha Women's and Children's Hospital Sokoto) and Wamakko (Orthopedic Hospital) LGA were selected for this study (Figure 1). Nasal swabs were collected randomly from healthcare workers, inpatients, outpatients, security guards and janitors. A structured proforma pertaining to demography and risk factors for S. aureus and MRSA colonization was discussed with participants (voluntarily) before sample collection. Samples were collected only from participants who gave informed consent. The right and left nostrils of consenting participants were swabbed using two different commercially available swab sticks. The swab was introduced into each nostril (premoistened with sterile normal saline) by rolling it 4 to 5 times (clockwise and anticlockwise) against the nostril wall [14]. Collected samples were then placed in a tube containing aqueous peptone solution, labeled, stored in icepacks and transported immediately to the central research laboratory for further processing.

Phenotypic screening to identify Staphylococcus aureus
Upon arrival in the laboratory, nasal swabs were inoculated into mannitol salt agar and incubated for 18-24 hours at 37°C. Characteristic golden yellow colonies with yellowish background emerging from the overnight culture were noted as presumptive Staphylococcus aureus colonies. The colonies were further characterized by standard bacteriological procedures such as colony morphology, Gram reaction, catalase test, coagulase test and Microgen™ STAPH-ID kit [15]. The phenotypically characterized isolates were then inoculated into nutrient agar slants and stored until further characterization.

Antibiotic susceptibility testing (AST)
Phenotypically confirmed isolates of Staphylococcus aureus were suspended in brain-heart infusion broth and incubated at 35°C for 7 hours to attain a 0.5 MacFarland standard. The suspension was transferred into a glass cuvette, and the absorbance at 630 nm was determined and adjusted to 0.1. With the aid of a mono-channel micropipette, 1000 µl of the standardized inoculum was introduced into Mueller-Hinton agar (MHA) and spread throughout the media by means of a sterile glass rod and allowed to dry. Subsequently, the antimicrobial disks were placed aseptically using sterile forceps, allowed a prediffusion time of 1 hour and incubated for 24 hours at 37°C [16].  [16]. Zone of inhibition diameters were measured and entered real-time into the WHONET 5.6 software database, which then interpreted the results using CLSI 2018 break-points built into the system. The results were recorded as interpreted values, i.e., "R" (Resistant), "I" (Intermediate) and "S" (Sensitive) [17]. Multi-resistant strains were divided into MDR (multiple drug-resistant), XDR (extensively drug-resistant) and PDR (pan drug-resistant). MDR bacteria were defined as resistant to antibiotics in at least three different classes of antibiotics. XDR bacteria were characterized by their sensitivity to only three or fewer classes of antibiotics, and the PDR bacteria were resistant to all classes of antibiotics used in the study [18].

Oxacillin resistance screening agar base (ORSAB) test
A standardized suspension (0.5 McFarland) of S. aureus isolates was prepared and inoculated onto ORSAB medium pre-supplemented with 6 µg/ml oxacillin and 4% sodium chloride (ORSAB) to form a microbial lawn, which was incubated at 35°C for 24 hours. The emergent bluish colonies from the overnight cultures were considered methicillin resistant [19], [20].
Latex slide agglutination test for detection of penicillin-binding protein (PBP2a) Penicillin binding protein (PBP2a) latex agglutination test kit (Oxoid, UK) is a rapid latex agglutination test kit used to confirm successful isolation of MRSA by the detection of PBP2a in Staphylococcus isolates. The presence of PBP2a was determined according to the guideline provided by the manufacturer. Colonies from ORSAB medium symbolic of methicillin resistance were tested for the production of the PBP2a protein. The protein from resistant bacteria was extracted using two extraction reagents (DR0903 and DR0904). A loopful of organisms was placed into a 1.5 ml microcentrifuge tube with 4 drops of extraction reagent DR0903 and placed in a water bath set at 75°C. After 5 minutes, the tubes were removed and allowed to cool. A single drop of extraction reagent DR0904 was added to each tube, mixed well, and centrifuged at 3,000 rpm for five minutes. The supernatant was used for the test. The latex agglutination test was conducted on test cards provided with the kit [21]. The sample number was written in one circle and the other circle was labeled "control". The latex reagents were well mixed, and a drop of test latex (DR0901) or control latex (DR0902) was added to their respectively labeled circles on the test card. 50 µL of the supernatant from the specimen was placed on the test circle and mixed with the mixing stick provided. The card was picked up and rocked back and forth for about three minutes while looking for agglutination under normal ambient light. The outcome of the test and the control reactions were noted visually. Agglutination of the test but not the control latex was considered positive, while no agglutination was considered negative [22].

Statistical analysis
The data were entered using SPSS version 25, their completeness and clearance were checked and then transferred to SAS, version 9.3 (SAS Institute Inc., Cary, North Carolina, USA). All the data were expressed in numbers and frequencies. Descriptive statistics was performed on the presence/absence of S. aureus and MRSA within the nares of each individual over the study period to determine the rate of carriage.

Modelling
Univariate associations of each risk factor with S. aureus and MRSA was examined using the frequency procedure of SAS, and a p-value ≤0.2 was considered significant. The risk factors found to be statistically significant by univariate logistic regression analysis at p≤0.2 were further evaluated by multivariate (forward selection procedure) regression model (p≤0.05) for associations with S. aureus and other coagulase positive staphylococci. A binary logistic regression model was used determine the association of the risk factor with MRSA. and p≤0.05 at a 95% CI was considered statistically significant. By using regression models, Adjusted Odds ratios (AOR) and 95% CI were calculated to identify how variables associated independently with the development of MRSA or S. aureus colonization. The primary analysis finally compared participants in whom MRSA colonization had occurred with those without MRSA colonization using oneway analysis of variance (ANOVA) ( Figure 2). The factors that fit the model appeared the most statistically plausible.      Figure 4B illustrates that most of the recovered coagulase-positive staphylococcal isolates were from participants at Specialist Hospital Sokoto. These isolates included S. aureus with 33 (40.7%), S. intermedius with   The relationship between the zones of inhibition of cefoxitin and linezolid is depicted in the scatterplot in Figure 6, which shows that 23.5% of isolates were resistant to both linezolid and cefoxitin, 28.4% were linezolid susceptible and cefoxitin resistant, and 48.1% of the isolates were susceptible to both linezolid and cefoxitin. The unpopulated area of the plot reflects the fact that there is no cefoxitin-susceptible isolate that was linezolid resistant. The doughnut chart in Figure 4A displays the overall prevalence of S. aureus (61.8%) after phenotypic characterization. As illustrated in Figure 4B, the percentage of S. aureus that is MRSA is 46.9% (38/81). The figure also shows the distribution of MRSA among the study centers. The relationships between MRSA and associated risk factors are shown in  Figure 7A shows the individual percentage distribution of multidrug-resistant Staphylococcus aureus (MDRSA), methicillin-susceptible S. aureus (MSSA), methicillin-resistant S. aureus (MRSA), extreme-   Figure 7B . Out of 74 participants, 7 (9.4) were dual-nostril carriers of S. aureus, out of which 3 (42.9%) were MRSA carriers. Table 6 shows that out of 116 participants who harboured coagulase positive S. aureus, 16 (13.7%) were dual-nostril carriers.
The results for the multivariate analysis of demographic and risk factors for Staphylococcus aureus and MRSA nasal colonization is presented in Table 7.

Discussion
Methicillin-resistant Staphylococcus aureus (MRSA) is a major human pathogen and a leading cause of nosocomial infections [8]. Recommendation in hospital surveillance protocols disagree as to whether both nostrils should be sampled, and those that favor the sampling of both nostrils habitually recommend the use of a single swab [23]. Implicit in such protocols is the idea that nostrils are homogenous regarding the bacterial populations they carry, thus justifying treating two separate nostrils as if they were a single body site. This study tested this premise by sampling right and left nostrils separately for S. aureus carriage. This study focused on S. aureus and MRSA isolates from the anterior nares of participants from three hospitals in Sokoto state. We documented carriage rates of 61.8% and 46.9% for S. aureus and MRSA in this study. These observed rates are consistent with the burden of S. aureus being higher and MRSA being lower than 50% prevalence in several African studies [3], [4], [5]. These reported rates may not be unconnected to non-adherence to drug prescription, self-medication, and poor hygiene practices coupled with suboptimal sanitation and water supply facilities in African countries [3], [24], [25]. . This may be because the hospital serves numerous patients from within and outside the metropolis, including referred patients from the other two health facilities. Epidemiologically, hospitals are reported to be connected by the patients they share, and their degree of connectedness influences the rates of spread of hospital-acquired bacteria [26].
Overall, the category of participants with the highest carriage rate was healthcare workers, probably because of greater exposure to circulating microbes [27]. Healthcare workers (HCWs) are more often colonized, serving as a reservoir for endogenous infections and dissemination [28]. This is corroborated by the 31% prevalence rate reported among health-care workers by other authors [29]. Sixty-three percent (63%; 95%, CI: 37.7-57.1) participants in this study were cultured-positive for S. aureus in the right nostril compared to 37% (95%, CI: 20. 2-42.8) in the left nostril. In total, 74 participants carried 81 S. aureus in their nostrils, out of which only 7 (9.5%, n=7/74) participants were dual-nostril carriers, and in turn, only 3 (42.9%, n=3/7) of the latter were dual carriers for MRSA. Kildow et al. [30] reported that nasal carriers of S. aureus were 60% likely to harbour the bacteria in a single nostril than in both (test of population proportions, P=0.0015). The participants in this study were more likely to carry more S. aureus in the right nostril than in the left (P=0.0010). In terms of MRSA, a significant association (P=0.0062) was recorded between right-and left-nostril carriage. Dual-nostril (MRSA) carriage, however, was less probable among participants than was single-nostril carriage (P=0.0001). This may be attributable to cyclic events known as the nasal cycle, where nasal airflow is greater in one nostril than in the other due to transient asymmetric nasal passage obstruction by swollen tissue. This physically blocks the passage of air in one nostril more than in the other, resulting in an increase in the filling pressure and other pathophysiological (e.g., nasal congestion) phenomena which favor higher bacterial adherence to the nasal epithelium [31]. In this study, the percentage carriage of S. aureus and MRSA was higher among the female participants. Female sex hormones (estrogen) have been related to high nasal S. aureus carriage. Over the past two decades, studies [32] have related high estrogen levels as the impetus for a shift in S. aureus carriage status. High estrogen levels could have predisposed the participants to higher colonization by altering the properties of nasal surfaces that serve as barriers to infection (e.g., local mucosal atrophy and decreased mucin secretion). This trend agrees with the findings of Liu et al. [32].  [37]. HIV-infected patients are at greater risk (poor immunity, exposure to antibiotics from recent hospitalizations and earlier MRSA infection or colonization) of MRSA colonization relative to the general population [38], [39]. Nevertheless, it was interesting that nasal colonization with MRSA was more often found in respondents without HIV/AIDS in our study. In fact, reduced MRSA colonization has been reported among HIV/AIDS patients on highly active antiretroviral therapy (HAART) by some studies [40], [41]. This unintended effect of antiretroviral drugs could help explain our findings. The reduced MRSA colonization may be linked to non-selection of drug-resistant microorganisms subsequent to reduced antibiotic usage among patients on HAART treatment [42]. Furthermore, a decreased HIV disease progression in patients undergoing therapy may have led to a reduced frequency of S. aureus and MRSA colonization [39], [43].
Regarding tobacco use, this study found that the colonization rate of S. aureus was higher among non-users of tobacco ( This includes the intrinsic nature of some isolates to be resistant to the drug, either due to lack of the target site for that drug, inability of the drug to transit through the organism cell wall or membrane and reach its site of action, acquisition of plasmids which is most common among hospital isolates, drug efflux and target site modification or permeability [53]. Ceftazidime was the most prevalent isolate (60.5%, n=49, 95% CI; 55.3-76.5), followed by cefoxitin (53.1%, n=43, 95% CI; 41.7-64.2). Gentamycin is the only drug in this study that was most effective against S. aureus. One reason for its effectiveness might be associated with the fact that gentamycin is infrequently used, as it administered by injection. This form of administration is far less amenable to self-medication than orally administered antibiotics [54]. Reports have documented that resistance to cefoxitin shown by disk diffusion can be used for MRSA strain detection in routine testing, because cefoxitin is regarded as a potential inducer of the system that regulates mecA gene [55]. For this reason, isolates which were found to be resistant to cefoxitin were also considered methicillin resistant. Overall, linezolid was effective (76.5%) across the study centers, although its resistance percentage was still 23.5% (95%CI; 16.1-35.8). Its effectiveness may be connected to the fact that, as opposed to the other antibiotics used in this study, linezolid is not routinely prescribed or administered. Conversely, resistance to linezolid observed in this study may have been mediated through ribosomal mutations (23SrRNA) or methylation of 23SrRNA by the horizontally transferred Cfr plasmidborne ribosomal methyltransferase [56]. The increased incidence of multi-resistant strains observed in this study may be due to the included study centres being secondary healthcare centres, and patients from adjoining districts and even villages are admitted for treatment. Before admission to the hospital, most of the participants may have taken different antibiotics prescribed by general practitioners or purchased overthe-counter, often in improper doses [57]. There are several limitations to this study. First, molecular differentiation of the strains into community and hospitalacquired (CA and HA) strains was not carried out to further determine the directionality of their spread. Second, the use of nasal cultures solely for S. aureus and MRSA detection has been reported to have a sensitivity of 78% to 85% which is much higher than cultures from other body sites [58], [59]. The sensitivity of our sampling technique was not accounted for during sample size determination.
Thus, our reported rates may not give a full account of the situation in the region. Nevertheless, our results revealed that MRSA colonization among the studied respondents occurred.

Conclusions
This study found a prevalence of 61.8% and 46.9% of S. aureus and MRSA among the studied hospitals in Sokoto state, indicating that the nares of the hospital populace are not free from S. aureus and MRSA colonization. Especially the fact that MRSA colonizes nostrils of participants (associated with risk factors HIV, marital status, diabetes, and tobacco usage) did not occur by chance. In total, 74 participants carried 81 S. aureus in their nostrils, out of which only 7 (9.5%, n=7/74) participants were dual-nostril carriers. Among these, only 3 (42.9%, n=3/7) were dual carriers for MRSA. Nasal carriers of S. aureus were significantly more likely to carry in one nostril than in both (test of population proportions, P=0.0001). A majority of the isolates were resistant to multiple antibiotics. Resistance was predominantly to ceftazidime and cefoxitin. In light of the findings in this study, we recommend that nasal swabs should be taken from the right and left nostrils of patients using two different commercially available swab sticks, as carriage in both nostrils is not homogeneous, i.e., each nostril should be treated as a separate anatomical site to avoid underestimation of MRSA carriage. Another method of swabbing both nostrils with one swab stick starting from the left may also prove effective in determining MRSA carrier status of respondents. We need to update our healthcare workers, especially physicians, microbiologists, infection control practitioners and bureaucrats managing health care about MRSA emergence and spread.

Competing interests
The authors declare that they have no competing interests.